65-kDa hsp-reactive T cells in NTX mice
Eur. J. Immunol. 1991. 21: 597-603
597
Akinori Iwasakiov, Yasunobu YoshikaiO, Hiroyuki YuukiO, Hiroaki TakimotoO and Kikuo NomotoO
Self-reactive T cells are activated by the 65-kDa mycobacterial heat-shock protein in neonatally thymectomized mice*
Department of Immunologyo, Medical Institute of Bioregulation, Kyushu University and Department of Microbiologyv, Fukuoka University School of Medicine, Fukuoka
To elucidate the mechanism of autoimmune disease in neonatally thymectomized (NTX) mice, we have investigated the responsiveness of the self-reactive Tcells which have not undergone clonal deletion in such animals. Consistent with a recent report (Yuuki et al., Eur. J. Zmmunol. 1990. 20: 1475), T cells bearing Vgll-gene products capable of recognizing I-E-encoded molecules were readily detected in the mature T cell pool of NTX BALB/c (I-Ed, M I s - ~ ~mice. ) The Vgll-bearing T cells in NTX mice expressed interleukin 2 receptors and responded normally to signals delivered through the T cell receptor. Notably, theseTcells in NTX mice proliferated significantly after culture with the 65-kDa mycobacterial heat-shock protein, whose amino acid sequence is highly homologous to that in eukaryotes.These results suggest that self-reactiveTcells in NTX mice may be activated by heat-shock proteins derived from various pathogens andor stressed autologous cells, resulting in the development of autoimmune diseases in such animals.
1 Introduction
Heat-shock protein (hsp), polypeptides phylogenetically conserved between prokaryotes, are frequently the target Tcell precursors derived from fetal liver or BM proliferate of humoral and cell-mediated immune responses during and differentiate in the thymus. During T cell differentia- infection and autoimmune diseases [13]. Among the vartion in the thymus, thymocytes with a range of affinities for ious kinds of hsp, the 65-kDa mycobacterial hsp has been self MHC from high to low are positively selected and identified as a target of Tcells capable of causing autoimthymocytes with high affinities for self antigens are toler- mune disease in a rat model of adjuvant-induced arthritis ized (reviewed in [l, 21). There are several lines of [14] and in the synovial infiltrates of rheumatoid arthritis convincing evidence that self tolerance is due mainly to the patiants [15]. We have previously reported that 65-kDa deletion of T cells bearing TcR capable of recognizing the hsp-reactiveTcellsbearingTcR y/S play an important role in self antigen in the thymus. It has been reported that Tcells the induction of autoimmune diseases in aged nude mice bearing Vpll or Vpl7a gene products capable of recogniz- bred under conventional condition [ 161. Multiple organing ligands of I-E are eliminated from mature T cell specific autoimmune diseases can be readily induced in repertoire of mice carrying I-E alleles [3-51. TcR using Vp3 NTX mice. It is not elucidated, however, whether the or Vp6B.l are also found to be specific for the products of self-reactiveTcells present in such animals are responsible M I s - ~or~ Mls-la and mice expressing Mls-la or M I s - ~ ~for the autoimmune diseases. eliminate Vp6/8.1- or V 3-bearing Tcells in their mature T cell pool, respectively 86-11]. On the other hand, recent In the present study, we have investigated the fate of work with aged athymic nude mice and neonatally thymec- self-reactive T cells and their responsiveness in BALB/c tomized (NTX) mice have revealed that self-reactiveTcells mice which had undergone neonatal thymectomy at day 3 are present in the matureTcell pool differentiatingalong an after birth, by assessing Vpll-bearing T cells reactive to I-E-encoded molecules. Our results indicate that the extrathymic pathway [12]. self-reactiveVpll-bearing Tcells are readily detected in the LN cells and can respond normally to signals delivered through the TcR in the presence of exogenous IL 2. Notably,we have found that the LN Tcells in NTX mice can produce an increased level of IL 2 in response to the 65-kDa [I 89001 mycobacterial hsp and theVpll-bearingTcells in these mice * This work was supported by grants to Y. Yoshikai from the proliferate significantly after culture with the 65-kDa hsp. Ministry of Education, Science and Culture, the Ministry of The implication of these findings for the development of Welfare and from Special Coordination Funds of the Science and autoimmune disease is discussed. TechnologyAgency of the Japanese government. This work also received financial support from the UNDPIWorld'BanklWHO Special Program for Research and Training in Tropical Disease (TDR). Correspondence: YasunobuYoshikai,Department of immunology,
Medical Institute of Bioregulation, Kyushu University, 3-1-1, Maidashi, Higashi-ku, Fukuoka 812, Japan Abbreviations: NTX: Newborn thymectomized hsp: Heatshock protein SE: Staphylococcal enterotoxin(s) 0 VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1991
2 Materials and methods 2.1 Mice
Female 7- to 8-week-old BALB/c and C57BL/6 mice were obtained from Japan SLC, (Shizuoka, Japan). Female and male BALB/c mice were mated in our laboratory, and 3-day-old female BALB/c mice anestetized by hypothermia
+
0014-2980/91/0303-0597$3.50 .25/0
598
Eur. J. Imrnunol. 1991. 21: 597-603
A. Iwasaki,Y. Yoshikai, H . Yuuki et al.
were thymectomized by the suction technique [17]. Mice sham-operated at 3 days after birth were used as controls. 2.2 Antibodies
The following antibodies were used for immunofluorescence staining: anti-CD3 mAb (145-2C11) kindly provided by Dr. J. A . Bluestone (University of Chicago), anti-Vg3 mAb (KJ25), and anti-Vg8.1 8.2 mAb (KJ-16), kindly provided by Drs. J. Kappler and I? Marrack (National Jewish Center for Immunology and Respiratory Medicine, Denver, Co), anti-Vg6 mAb (44-22-1) kindly provided by Dr. H. Hengartner (University Hospital, Zurich), antiVgll mAb (KT11) kindly provided by Dr.Tomonari (MRC Clinical Research Center), anti-TcR/a/P mAb (H57-597) kindly provided by Dr. R. A. Kubo (National Jewish Center Immunology and Respiratory Medicine) , PEconjugated anti-mouse CD4 mAb (Becton Dickinson, Mountain View, CA), biotin-conjugated anti-IL 2R (7D4), or biotin-conjugated anti-mouse CD8 mAb, PE-conjugated streptavidin and streptavidin-DuoCHROME purchased from Becton Dickinson, PE-conjugated antiThy-1.2 purchased from Caltag Laboratories Inc. (San Francisco, CA), FITC-conjugated goat anti-rat IgG and FITC-conjugated goat anti-hamster IgG purchased from Tag0 Inc. (Burlingame, CA).
+
2.3 Staphylococcal enterotoxin A (SEA) SEA was isolated and prepared by the methods of Oda [ 181. SEA derived from Staphylococcus aureus 13N-2909 was kindly provided by the Tokyo Metropolitan Research of Public Health through the courtesy of Dr. H. Igarashi.
with FITC-conjugated goat anti-rat IgG after treatment with anti-Vg6 mAb, or anti-Vgll mAb and PE-conjugated anti-Thy-1.2 mAb or biotin-conjugated anti-IL 2R plus streptavidin-DuoCHROME. The LN cells (1 x W m l ) were cultured with the 65-kDa hsp (25 pg/ml) and 3 days later, the responding cells were stained with anti-Vg3 mAb, anti-Vg6 mAb, anti-Vg8.1 + 8.2 mAb, anti-Vgll mAb plus FITC-anti-hamster IgG or rat IgG and PE-anti-Thy-1.2 mAb, or FITC-anti-CD8 mAb plus PE-anti-CD4 mAb and analyzed by FACScan (Becton Dickinson).
2.6 Assay for [3H]dThd incorporation and 1L 2 production of T cells in response to the 65-kDa hsp LN cells were stimulated with various concentrationsof the 65-kDa recombinant hsp derived from Mycobacteriurn bovis (kindly provided by Drs. R.Van der Zee, and Dr. J. D. A. Van Embden, National Institute of Public Health and Environment Protection, Bilthoven, Netherlands) in 0.2 mi medium at 37 "Cin a CO2 incubator for 72 h in RPMI 1640 with FCS, and then 37 kBq (= 1 pCi) [3H]dThdwas added to each well. Cultures were incubated for another 6 h and the responder cells were harvested with a cell harvester (Wallac, 1295, LKB, Turku Finland). [3H]dThd incorporation by respondercells was measured in a liquid scintillation counter (Wallac, 1205). For IL 2 production, CTLL-2 cells (1 x 104, IL 2-dependent cell line) were incubated with a diluted SN from cultures in which LN cells were stimulated with the 65-kDa hsp in 0.2 ml medium at 37 "Cfor 48 h, and then, 37 kBq [3H]dThd was added to each well. Cultures were incubated for another 6 hand [3H]dThdincorporation by responder cells was measured in a liquid scintillation counter (LKB). 2.7 Titer of anti-DNA antibody in serum
2.4 FCM analysis Spleen and LN cells were taken from sham-operated BALBlc or NTX mice at 5 months of age. A single-cell suspension was stained with FITC-conjugated anti-mouse CD3 mAb.The LN cells were also stained with anti-TcR/a/P mAb or each anti-Vg mAb plus FITC-conjugated antihamster or rat IgG and PE-conjugated anti-Thy-1.2 mAb, and then analyzed on a FACScan flow cytometer (Becton Dickinson). The LN cells of the NTX mice were stained with anti-Vg6 mAb or anti-Vpll mAb plus FITC-anti-rat IgG mAb and PE-anti-Thy-1.2 mAb, biotin-anti41 2R mAb plus streptavidin-DuoCHROME.The percentages of fluorescent cells were determined by integration from profiles based on 3 x 104 cells.
Anti-DNA titers in the serum were determined by an ELISA [16, 191. 2.8 Statistics
The statical significance of the data was determined by the Student's t-test. A p value of
Eur. J. Immunol. 1991. 21: 597-603
597
Akinori Iwasakiov, Yasunobu YoshikaiO, Hiroyuki YuukiO, Hiroaki TakimotoO and Kikuo NomotoO
Self-reactive T cells are activated by the 65-kDa mycobacterial heat-shock protein in neonatally thymectomized mice*
Department of Immunologyo, Medical Institute of Bioregulation, Kyushu University and Department of Microbiologyv, Fukuoka University School of Medicine, Fukuoka
To elucidate the mechanism of autoimmune disease in neonatally thymectomized (NTX) mice, we have investigated the responsiveness of the self-reactive Tcells which have not undergone clonal deletion in such animals. Consistent with a recent report (Yuuki et al., Eur. J. Zmmunol. 1990. 20: 1475), T cells bearing Vgll-gene products capable of recognizing I-E-encoded molecules were readily detected in the mature T cell pool of NTX BALB/c (I-Ed, M I s - ~ ~mice. ) The Vgll-bearing T cells in NTX mice expressed interleukin 2 receptors and responded normally to signals delivered through the T cell receptor. Notably, theseTcells in NTX mice proliferated significantly after culture with the 65-kDa mycobacterial heat-shock protein, whose amino acid sequence is highly homologous to that in eukaryotes.These results suggest that self-reactiveTcells in NTX mice may be activated by heat-shock proteins derived from various pathogens andor stressed autologous cells, resulting in the development of autoimmune diseases in such animals.
1 Introduction
Heat-shock protein (hsp), polypeptides phylogenetically conserved between prokaryotes, are frequently the target Tcell precursors derived from fetal liver or BM proliferate of humoral and cell-mediated immune responses during and differentiate in the thymus. During T cell differentia- infection and autoimmune diseases [13]. Among the vartion in the thymus, thymocytes with a range of affinities for ious kinds of hsp, the 65-kDa mycobacterial hsp has been self MHC from high to low are positively selected and identified as a target of Tcells capable of causing autoimthymocytes with high affinities for self antigens are toler- mune disease in a rat model of adjuvant-induced arthritis ized (reviewed in [l, 21). There are several lines of [14] and in the synovial infiltrates of rheumatoid arthritis convincing evidence that self tolerance is due mainly to the patiants [15]. We have previously reported that 65-kDa deletion of T cells bearing TcR capable of recognizing the hsp-reactiveTcellsbearingTcR y/S play an important role in self antigen in the thymus. It has been reported that Tcells the induction of autoimmune diseases in aged nude mice bearing Vpll or Vpl7a gene products capable of recogniz- bred under conventional condition [ 161. Multiple organing ligands of I-E are eliminated from mature T cell specific autoimmune diseases can be readily induced in repertoire of mice carrying I-E alleles [3-51. TcR using Vp3 NTX mice. It is not elucidated, however, whether the or Vp6B.l are also found to be specific for the products of self-reactiveTcells present in such animals are responsible M I s - ~or~ Mls-la and mice expressing Mls-la or M I s - ~ ~for the autoimmune diseases. eliminate Vp6/8.1- or V 3-bearing Tcells in their mature T cell pool, respectively 86-11]. On the other hand, recent In the present study, we have investigated the fate of work with aged athymic nude mice and neonatally thymec- self-reactive T cells and their responsiveness in BALB/c tomized (NTX) mice have revealed that self-reactiveTcells mice which had undergone neonatal thymectomy at day 3 are present in the matureTcell pool differentiatingalong an after birth, by assessing Vpll-bearing T cells reactive to I-E-encoded molecules. Our results indicate that the extrathymic pathway [12]. self-reactiveVpll-bearing Tcells are readily detected in the LN cells and can respond normally to signals delivered through the TcR in the presence of exogenous IL 2. Notably,we have found that the LN Tcells in NTX mice can produce an increased level of IL 2 in response to the 65-kDa [I 89001 mycobacterial hsp and theVpll-bearingTcells in these mice * This work was supported by grants to Y. Yoshikai from the proliferate significantly after culture with the 65-kDa hsp. Ministry of Education, Science and Culture, the Ministry of The implication of these findings for the development of Welfare and from Special Coordination Funds of the Science and autoimmune disease is discussed. TechnologyAgency of the Japanese government. This work also received financial support from the UNDPIWorld'BanklWHO Special Program for Research and Training in Tropical Disease (TDR). Correspondence: YasunobuYoshikai,Department of immunology,
Medical Institute of Bioregulation, Kyushu University, 3-1-1, Maidashi, Higashi-ku, Fukuoka 812, Japan Abbreviations: NTX: Newborn thymectomized hsp: Heatshock protein SE: Staphylococcal enterotoxin(s) 0 VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1991
2 Materials and methods 2.1 Mice
Female 7- to 8-week-old BALB/c and C57BL/6 mice were obtained from Japan SLC, (Shizuoka, Japan). Female and male BALB/c mice were mated in our laboratory, and 3-day-old female BALB/c mice anestetized by hypothermia
+
0014-2980/91/0303-0597$3.50 .25/0
598
Eur. J. Imrnunol. 1991. 21: 597-603
A. Iwasaki,Y. Yoshikai, H . Yuuki et al.
were thymectomized by the suction technique [17]. Mice sham-operated at 3 days after birth were used as controls. 2.2 Antibodies
The following antibodies were used for immunofluorescence staining: anti-CD3 mAb (145-2C11) kindly provided by Dr. J. A . Bluestone (University of Chicago), anti-Vg3 mAb (KJ25), and anti-Vg8.1 8.2 mAb (KJ-16), kindly provided by Drs. J. Kappler and I? Marrack (National Jewish Center for Immunology and Respiratory Medicine, Denver, Co), anti-Vg6 mAb (44-22-1) kindly provided by Dr. H. Hengartner (University Hospital, Zurich), antiVgll mAb (KT11) kindly provided by Dr.Tomonari (MRC Clinical Research Center), anti-TcR/a/P mAb (H57-597) kindly provided by Dr. R. A. Kubo (National Jewish Center Immunology and Respiratory Medicine) , PEconjugated anti-mouse CD4 mAb (Becton Dickinson, Mountain View, CA), biotin-conjugated anti-IL 2R (7D4), or biotin-conjugated anti-mouse CD8 mAb, PE-conjugated streptavidin and streptavidin-DuoCHROME purchased from Becton Dickinson, PE-conjugated antiThy-1.2 purchased from Caltag Laboratories Inc. (San Francisco, CA), FITC-conjugated goat anti-rat IgG and FITC-conjugated goat anti-hamster IgG purchased from Tag0 Inc. (Burlingame, CA).
+
2.3 Staphylococcal enterotoxin A (SEA) SEA was isolated and prepared by the methods of Oda [ 181. SEA derived from Staphylococcus aureus 13N-2909 was kindly provided by the Tokyo Metropolitan Research of Public Health through the courtesy of Dr. H. Igarashi.
with FITC-conjugated goat anti-rat IgG after treatment with anti-Vg6 mAb, or anti-Vgll mAb and PE-conjugated anti-Thy-1.2 mAb or biotin-conjugated anti-IL 2R plus streptavidin-DuoCHROME. The LN cells (1 x W m l ) were cultured with the 65-kDa hsp (25 pg/ml) and 3 days later, the responding cells were stained with anti-Vg3 mAb, anti-Vg6 mAb, anti-Vg8.1 + 8.2 mAb, anti-Vgll mAb plus FITC-anti-hamster IgG or rat IgG and PE-anti-Thy-1.2 mAb, or FITC-anti-CD8 mAb plus PE-anti-CD4 mAb and analyzed by FACScan (Becton Dickinson).
2.6 Assay for [3H]dThd incorporation and 1L 2 production of T cells in response to the 65-kDa hsp LN cells were stimulated with various concentrationsof the 65-kDa recombinant hsp derived from Mycobacteriurn bovis (kindly provided by Drs. R.Van der Zee, and Dr. J. D. A. Van Embden, National Institute of Public Health and Environment Protection, Bilthoven, Netherlands) in 0.2 mi medium at 37 "Cin a CO2 incubator for 72 h in RPMI 1640 with FCS, and then 37 kBq (= 1 pCi) [3H]dThdwas added to each well. Cultures were incubated for another 6 h and the responder cells were harvested with a cell harvester (Wallac, 1295, LKB, Turku Finland). [3H]dThd incorporation by respondercells was measured in a liquid scintillation counter (Wallac, 1205). For IL 2 production, CTLL-2 cells (1 x 104, IL 2-dependent cell line) were incubated with a diluted SN from cultures in which LN cells were stimulated with the 65-kDa hsp in 0.2 ml medium at 37 "Cfor 48 h, and then, 37 kBq [3H]dThd was added to each well. Cultures were incubated for another 6 hand [3H]dThdincorporation by responder cells was measured in a liquid scintillation counter (LKB). 2.7 Titer of anti-DNA antibody in serum
2.4 FCM analysis Spleen and LN cells were taken from sham-operated BALBlc or NTX mice at 5 months of age. A single-cell suspension was stained with FITC-conjugated anti-mouse CD3 mAb.The LN cells were also stained with anti-TcR/a/P mAb or each anti-Vg mAb plus FITC-conjugated antihamster or rat IgG and PE-conjugated anti-Thy-1.2 mAb, and then analyzed on a FACScan flow cytometer (Becton Dickinson). The LN cells of the NTX mice were stained with anti-Vg6 mAb or anti-Vpll mAb plus FITC-anti-rat IgG mAb and PE-anti-Thy-1.2 mAb, biotin-anti41 2R mAb plus streptavidin-DuoCHROME.The percentages of fluorescent cells were determined by integration from profiles based on 3 x 104 cells.
Anti-DNA titers in the serum were determined by an ELISA [16, 191. 2.8 Statistics
The statical significance of the data was determined by the Student's t-test. A p value of
